Essential hypertension causes severe complications such as apoplexy, ischemic heart diseases and nephrosclerosis. These complications are fundamentally associated with vascular disorders by excessive proliferation of vascular smooth muscle cells (VSMC), and are the targets of the treatment of hypertension. On the other hand, restenosis of coronary arteries takes place in about 40% after percutaneous transluminal coronary angioplasty (PTCA) as a treatment of angina pectoris and myocardial infarction, and further in about 30% after stent implantation. Histopathologically, TGF-β has been known to be involved in artery proliferative disorders such as hypertension vascular disease, neointimal formation after anigioplasty and in-stent neointimal formation, and atherosclerosis. Cell growth in such diseases can be inhibited by various action mechanisms, and one of them is to inhibit the expression of TGF-β.
Initially, TGF was found as a factor to change a normal cell to malignant one in mouse 3T3 cells that had been transformed by Molony sarcoma virus (MSV), and is roughly classified into TGF-α and TGF-β. TGF-β is a part composed of 112 amino acids at C-terminal side of a protein having a molecular weight of about 40,000 composed of 390 to 412 amino acids synthesized as a precursor, and that part forms a dimer (25 kDa) through disulfide-bond to have an activity.
TGF-β constitutes one family of protein that regulates the growth and development of cells (Non-patent Document 1). TGF-β is produced in various tissues such as blood vessel, platelet, liver, kidney, heart muscle, lung, pancreas, skin, placenta, and bone marrow, and has actions of cell growth, extracellular matrix formation, and immunity regulation.
TGF-β works on most cells to inhibit the growth, but has a biphasic growth action to mesenchymal cells such as fibroblast and vascular smooth muscle cell (VSMC). In other words, in these cells, TGF-β usually works to inhibit the growth but works to stimulate the growth when inflammation, mechanical stress or the like is given. These findings show that TGF-β is involved in neointimal formation after vascular injury by facilitating VSMC proliferation and extracellular matrix formation with increases in fibronectin and collagen. TGF-β is also involved in the formation of focus of arterial sclerosis. Based on the information, it is considered that local vascular therapy directed to the regulation of TGF-β effect is effective to alleviate the above vascular proliferative diseases.
Further, it is considered that TGF-β is involved in restenosis of renal artery after percutaneous transluminal renal artery angioplasty. From these facts, a TGF-β gene expression inhibition drug of the present invention is effective as a therapeutic agent to patients with the above various vascular proliferation/stenosis diseases.
Methods for inactivating gene functions by reverse genetics are used for analyzing a specific gene function, whereas such methods open high possibilities of therapy for other diseases based on virus infection, cancer, and abnormal genomic diseases. The inactivation of gene function can be performed at DNA level by homologous recombination or at RNA level by antisense oligodeoxynucleotide or ribozyme. However, methods using antisense oligodeoxynucleotide or ribozyme have drawbacks: limitation to target sequences; poor entry to tissues and cells; and easy degradation by nucleases.
On the other hand, unlike the nucleic acid medicines such as antisense oligodeoxynucleotide and ribozyme, it has been reported that pyrrole-imidazole polyamides (hereinafter also referred to as Py-Im polyamide) specifically recognize DNA base sequences and extracellularly control the expression of a specific gene.
Py-Im polyamides are a group of small synthetic molecules composed of the aromatic rings of the N-methylpyrrole unit (hereinafter also referred to as Py) and N-methylimidazole unit (hereinafter also referred to as Im) (Non-patent Documents 2 to 4). Py and Im are successively coupled to each other so that the Py-Im polyamide can be folded to have a U-shaped conformation in the presence of γ-aminobutyric acid. In a Py-Im of the present invention, an N-methylpyrrole unit (Py), an N-methylimidazole unit (Im), and a γ-aminobutyric acid unit (also referred to as γ linker) are bound to each other by amide bond (—C(═O)—NH—), and its general structure and production method are well known (see Patent Documents 1 to 3).
These synthetic Py-Im polyamides can bind to specific base pairs in a minor groove of double-stranded DNA with high affinity and specificity. Specific recognition of base pairs is dependent on one-to-one pair formation of Py and Im. That is, in U-shaped conformation in the minor groove of DNA, a Py/Im pair targets a CG base pair, Im/Py pair targets a GC base pair, and a Py/Py pair targets both AT and TA base pairs (Non-patent Documents 3 and 4). Recent studies have shown that the AT degeneracy can be overcome by replacing one pyrrole ring of the Py/Py pair with 3-hydroxypyrrole (Hp), and as a result of that, allowing a Hp/Py pair to preferentially bind to a T/A pair (Non-patent Document 5).
It is generally considered that transcription initiation is an important site for gene regulation. Initiation of transcription requires some transcription factors binding to specific recognition sequences in the gene promoter region. Py-Im polyamides designed to target the transcription factor binding site block the binding of the transcription factors and inhibit the gene expression, if the transcription factors are important for the gene expression. These principles have been proved by in vitro and in vivo experiments. An eight-ring Py-Im polyamide that bound in the recognition site of zinc finger (TFIIIA binding site) inhibited the transcription of 5S RNA genes (Non-patent Document 6). Polyamides that bind to base pairs adjacent to transcription factor sequences in a human immunodeficiency virus 1 type (HIV-1) promoter inhibits the replication of HIV-1 in a human cell. These sequences include a TATA box, a lymphoid enhancer factor LEF-1 sequence, and an ETS-1 sequence (Non-patent Document 7). In contrast, Py-Im polyamides also block a repressor factor or replace an inherent transcription factor, and thereby activate gene expression (Non-patent Documents 8 to 10). Human cytomegalovirus (CMV) UL122 mediate early protein 2 (IE86) blocks the replenishment of RNA polymerase II to a promoter to suppress the transcription of its related gene (Non-patent Document 8). Synthetic Py-Im polyamides block the suppression of IE86 and liberate the expression of the corresponding gene (Non-patent Document 9). A polyamide designed by Mapp et al. works as an artificial transcription factor and mediates gene transcription reaction (Non-patent Document 10).
Patent Document 1: Japanese Patent No. 3045706
Patent Document 2: JP-A-2001-136974
Patent Document 3: WO 03/000683 A1
Non-patent Document 1: Piek et al., FASEB J, 13, 2105-2124 (1999)
Non-patent Document 2: Trauger et al: Nature. 1996; 382: 559-61
Non-patent Document 3: White et al: Chem. Biol., 1997; 4: 569-78
Non-patent Document 4: Dervan: Bioorg Med. Chem. 2001; 9: 2215-35
Non-patent Document 5: White at al: Nature. 1998; 391: 468-71
Non-patent Document 6: Gottesfeld et al: Nature. 1997; 387: 202-5
Non-patent Document 7: Dickinson et al: Proc Natl Acad Sci USA. 1998; 95: 12890-5
Non-patent Document 8: Lee et al: Proc Natl Acad Sci USA. 1996; 93: 2570-5
Non-patent Document 9: Dickinson et al: Biochemistry. 1999; 38: 10801-7
Non-patent Document 10: Mapp et al: Proc Natl Acad Sci USA. 2000; 97: 3930-5